Research Article
Abstract
Cyclin I is an atypical cyclin because it is most abundant in postmitotic cells. We previously showed that cyclin I does not regulate proliferation, but rather controls survival of podocytes, terminally differentiated epithelial cells that are essential for the structural and functional integrity of kidney glomeruli. Here, we investigated the mechanism by which cyclin I safeguards against apoptosis and found that cyclin I bound and activated cyclin-dependent kinase 5 (Cdk5) in isolated mouse podocytes and neurons. Cdk5 activity was reduced in glomeruli and brain lysates from cyclin I–deficient mice, and inhibition of Cdk5 increased in vitro the susceptibility to apoptosis in response to cellular damage. In addition, levels of the prosurvival proteins Bcl-2 and Bcl-XL were reduced in podocytes and neurons from cyclin I–deficient mice, and restoration of Bcl-2 or Bcl-XL expression prevented injury-induced apoptosis. Furthermore, we found that levels of phosphorylated MEK1/2 and ERK1/2 were decreased in cyclin I–deficient podocytes and that inhibition of MEK1/2 restored Bcl2 and Bcl-XL protein levels. Of interest, this pathway was also defective in mice with experimental glomerulonephritis. Taken together, these data suggest that a cyclin I–Cdk5 complex forms a critical antiapoptotic factor in terminally differentiated cells that functions via MAPK signaling to modulate levels of the prosurvival proteins Bcl-2 and Bcl-XL.
Authors
Paul T. Brinkkoetter, Paul Olivier, Jimmy S. Wu, Scott Henderson, Ronald D. Krofft, Jeffrey W. Pippin, David Hockenbery, James M. Roberts, Stuart J. Shankland
Abstract
Although many self-reactive T cells are eliminated by negative selection in the thymus, some of these cells escape into the periphery, where they must be controlled by additional mechanisms. However, the molecular mechanisms underlying peripheral T cell tolerance and its maintenance remain largely undefined. In this study, we report that sirtuin 1 (Sirt1), a type III histone deacetylase, negatively regulates T cell activation and plays a major role in clonal T cell anergy in mice. In vivo, we found that loss of Sirt1 function resulted in abnormally increased T cell activation and a breakdown of CD4+ T cell tolerance. Conversely, upregulation of Sirt1 expression led to T cell anergy, in which the activity of the transcription factor AP-1 was substantially diminished. Furthermore, Sirt1 interacted with and deacetylated c-Jun, yielding an inactive AP-1 factor. In addition, Sirt1-deficient mice were unable to maintain T cell tolerance and developed severe experimental allergic encephalomyelitis as well as spontaneous autoimmunity. These findings provide insight into the molecular mechanisms of T cell activation and anergy, and we suggest that activators of Sirt1 may be useful as therapeutic agents for the treatment and/or prevention of autoimmune diseases.
Authors
Jinping Zhang, Sang-Myeong Lee, Stephen Shannon, Beixue Gao, Weimin Chen, An Chen, Rohit Divekar, Michael W. McBurney, Helen Braley-Mullen, Habib Zaghouani, Deyu Fang
Abstract
Inappropriate activation of developmental pathways is a well-recognized tumor-promoting mechanism. Here we show that overexpression of the homeoprotein Six1, normally a developmentally restricted transcriptional regulator, increases TGF-β signaling in human breast cancer cells and induces an epithelial-mesenchymal transition (EMT) that is in part dependent on its ability to increase TGF-β signaling. TGF-β signaling and EMT have been implicated in metastatic dissemination of carcinoma. Accordingly, we used spontaneous and experimental metastasis mouse models to demonstrate that Six1 overexpression promotes breast cancer metastasis. In addition, we show that, like its induction of EMT, Six1-induced experimental metastasis is dependent on its ability to activate TGF-β signaling. Importantly, in human breast cancers Six1 correlated with nuclear Smad3 and thus increased TGF-β signaling. Further, breast cancer patients whose tumors overexpressed Six1 had a shortened time to relapse and metastasis and an overall decrease in survival. Finally, we show that the effects of Six1 on tumor progression likely extend beyond breast cancer, since its overexpression correlated with adverse outcomes in numerous other cancers including brain, cervical, prostate, colon, kidney, and liver. Our findings indicate that Six1, acting through TGF-β signaling and EMT, is a powerful and global promoter of cancer metastasis.
Authors
Douglas S. Micalizzi, Kimberly L. Christensen, Paul Jedlicka, Ricardo D. Coletta, Anna E. Barón, J. Chuck Harrell, Kathryn B. Horwitz, Dean Billheimer, Karen A. Heichman, Alana L. Welm, William P. Schiemann, Heide L. Ford
Abstract
The mechanisms underlying mucus-associated pathologies in cystic fibrosis (CF) remain obscure. However, recent studies indicate that CF transmembrane conductance regulator (CFTR) is required for bicarbonate (HCO3–) transport and that HCO3– is critical for normal mucus formation. We therefore investigated the role of HCO3– in mucus secretion using mouse small intestine segments ex vivo. Basal rates of mucus release in the presence or absence of HCO3– were similar. However, in the absence of HCO3–, mucus release stimulated by either PGE2 or 5-hydroxytryptamine (5-HT) was approximately half that stimulated by these molecules in the presence of HCO3–. Inhibition of HCO3– and fluid transport markedly reduced stimulated mucus release. However, neither absence of HCO3– nor inhibition of HCO3– transport affected fluid secretion rates, indicating that the effect of HCO3– removal on mucus release was not due to decreased fluid secretion. In a mouse model of CF (mice homozygous for the most common human CFTR mutation), intestinal mucus release was minimal when stimulated with either PGE2 or 5-HT in the presence or absence of HCO3–. These data suggest that normal mucus release requires concurrent HCO3– secretion and that the characteristically aggregated mucus observed in mucin-secreting organs in individuals with CF may be a consequence of defective HCO3– transport.
Authors
Mary Abigail S. Garcia, Ning Yang, Paul M. Quinton
Abstract
Six1 is a developmentally regulated homeoprotein with limited expression in most normal adult tissues and frequent misexpression in a variety of malignancies. Here we demonstrate, using a bitransgenic mouse model, that misexpression of human Six1 in adult mouse mammary gland epithelium induces tumors of multiple histological subtypes in a dose-dependent manner. The neoplastic lesions induced by Six1 had an in situ origin, showed diverse differentiation, and exhibited progression to aggressive malignant neoplasms, as is often observed in human carcinoma of the breast. Strikingly, the vast majority of Six1-induced tumors underwent an epithelial-mesenchymal transition (EMT) and expressed multiple targets of activated Wnt signaling, including cyclin D1. Interestingly, Six1 and cyclin D1 coexpression was found to frequently occur in human breast cancers and was strongly predictive of poor prognosis. We further show that Six1 promoted a stem/progenitor cell phenotype in the mouse mammary gland and in Six1-driven mammary tumors. Our data thus provide genetic evidence for a potent oncogenic role for Six1 in mammary epithelial neoplasia, including promotion of EMT and stem cell–like features.
Authors
Erica L. McCoy, Ritsuko Iwanaga, Paul Jedlicka, Nee-Shamo Abbey, Lewis A. Chodosh, Karen A. Heichman, Alana L. Welm, Heide L. Ford
Abstract
Progressive familial heart block type I (PFHBI) is a progressive cardiac bundle branch disease in the His-Purkinje system that exhibits autosomal-dominant inheritance. In 3 branches of a large South African Afrikaner pedigree with an autosomal-dominant form of PFHBI, we identified the mutation c.19G→A in the transient receptor potential cation channel, subfamily M, member 4 gene (TRPM4) at chromosomal locus 19q13.3. This mutation predicted the amino acid substitution p.E7K in the TRPM4 amino terminus. TRPM4 encodes a Ca2+-activated nonselective cation (CAN) channel that belongs to the transient receptor potential melastatin ion channel family. Quantitative analysis of TRPM4 mRNA content in human cardiac tissue showed the highest expression level in Purkinje fibers. Cellular expression studies showed that the c.19G→A missense mutation attenuated deSUMOylation of the TRPM4 channel. The resulting constitutive SUMOylation of the mutant TRPM4 channel impaired endocytosis and led to elevated TRPM4 channel density at the cell surface. Our data therefore revealed a gain-of-function mechanism underlying this type of familial heart block.
Authors
Martin Kruse, Eric Schulze-Bahr, Valerie Corfield, Alf Beckmann, Birgit Stallmeyer, Güven Kurtbay, Iris Ohmert, Ellen Schulze-Bahr, Paul Brink, Olaf Pongs
Abstract
Although the modulation of ion channel gating by hormones and drugs has been extensively studied, much less is known about how cell surface ion channel expression levels are regulated. Here, we demonstrate that the cell surface density of both the heterologously expressed K+ channel encoded by the human ether-a-go-go–related gene (HERG) and its native counterpart, the rapidly activating delayed rectifier K+ channel (IKr), in rabbit hearts in vivo is precisely controlled by extracellular K+ concentration ([K+]o) within a physiologically relevant range. Reduction of [K+]o led to accelerated internalization and degradation of HERG channels within hours. Confocal analysis revealed colocalization between HERG and ubiquitin during the process of HERG internalization, and overexpression of ubiquitin facilitated HERG degradation under low [K+]o. The HERG channels colocalized with a marker of multivesicular bodies during internalization, and the internalized HERG channels were targeted to lysosomes. Our results provide the first evidence to our knowledge that the cell surface density of a voltage-gated K+ channel, HERG, is regulated by a biological factor, extracellular K+. Because hypokalemia is known to exacerbate long QT syndrome (LQTS) and Torsades de pointes tachyarrhythmias, our findings provide a potential mechanistic link between hypokalemia and LQTS.
Authors
Jun Guo, Hamid Massaeli, Jianmin Xu, Zongchao Jia, Jeffrey T. Wigle, Nasrin Mesaeli, Shetuan Zhang
Abstract
VSMCs respond to changes in the local environment by adjusting their phenotype from contractile to synthetic, a phenomenon known as phenotypic modulation or switching. Failure of VSMCs to acquire and maintain the contractile phenotype plays a key role in a number of major human diseases, including arteriosclerosis. Although several regulatory circuits that control differentiation of SMCs have been identified, the decisive mechanisms that govern phenotypic modulation remain unknown. Here, we demonstrate that the mouse miR-143/145 cluster, expression of which is confined to SMCs during development, is required for VSMC acquisition of the contractile phenotype. VSMCs from miR-143/145–deficient mice were locked in the synthetic state, which incapacitated their contractile abilities and favored neointimal lesion development. Unbiased high-throughput, quantitative, mass spectrometry–based proteomics using reference mice labeled with stable isotopes allowed identification of miR-143/145 targets; these included angiotensin-converting enzyme (ACE), which might affect both the synthetic phenotype and contractile functions of VSMCs. Pharmacological inhibition of either ACE or the AT1 receptor partially reversed vascular dysfunction and normalized gene expression in miR-143/145–deficient mice. We conclude that manipulation of miR-143/145 expression may offer a new approach for influencing vascular repair and attenuating arteriosclerotic pathogenesis.
Authors
Thomas Boettger, Nadine Beetz, Sawa Kostin, Johanna Schneider, Marcus Krüger, Lutz Hein, Thomas Braun
Abstract
Pharmacologic blockade of EGFR or the closely related receptor ERBB2 has modest efficacy against colorectal cancers in the clinic. Although the upregulation of ERBB3, a pseudo-kinase member of the EGFR/ERBB family, is known to contribute to EGFR inhibitor resistance in other cancers, its functions in normal and malignant intestinal epithelium have not been defined. We have shown here that the intestinal epithelium of mice with intestine-specific genetic ablation of Erbb3 exhibits no cytological abnormalities but does exhibit loss of expression of ERBB4 and sensitivity to intestinal damage. By contrast, intestine-specific Erbb3 ablation resulted in almost complete absence of intestinal tumors in the ApcMin mouse model of colon cancer. Unlike nontransformed epithelium lacking ERBB3, intestinal tumors lacking ERBB3 had reduced PI3K/AKT signaling, which led to attenuation of tumorigenesis via a tumor-specific increase in caspase-3–mediated apoptosis. Consistent with the mouse data, which suggest that ERBB3-ERBB4 heterodimers contribute to colon cancer survival, experimentally induced loss of ERBB3 in a KRAS mutant human colon cancer cell line was associated with loss of ERBB4 expression, and siRNA knockdown of either ERBB3 or ERBB4 resulted in elevated levels of apoptosis. These results indicate that the ERBB3 pseudo-kinase has essential roles in supporting intestinal tumorigenesis and suggest that ERBB3 may be a promising target for the treatment of colorectal cancers.
Authors
Daekee Lee, Ming Yu, Eunjung Lee, Hyunok Kim, Yanan Yang, Kyoungmi Kim, Christina Pannicia, Jonathan M. Kurie, David W. Threadgill
Abstract
Genetic instability, which leads to an accumulation of various genetic abnormalities, has been considered an essential component of the human neoplasic transformation process. However, the molecular basis of genomic instability during tumorigenesis remains incompletely understood. Growing evidence indicates that checkpoint with forkhead and ring finger domains (CHFR), a recently identified mitotic checkpoint protein, plays an important role in maintaining chromosome integrity and functions as a tumor suppressor. In this study, we used high-throughput technology to conduct gene expression profiling of human colon cancers and found that loss of CHFR expression frequently occurred in colon cancers with high microsatellite instability (MSI-H). Downregulation of CHFR expression was closely associated with overexpression of Aurora A, an important mitotic kinase. Mice with deficiencies in both Chfr and Mlh1 (the gene that encodes the DNA mismatch-repair protein Mlh1) displayed dramatically higher incidence of spontaneous tumors relative to mice deficient for only one of these genes. These results suggest that defects in both Chfr and Mlh1 synergistically increase predisposition to tumorigenesis.
Authors
Zheng Fu, Kevin Regan, Lizhi Zhang, Michael H. Muders, Stephen N. Thibodeau, Amy French, Yanhong Wu, Scott H. Kaufmann, Wilma L. Lingle, Junjie Chen, Donald J. Tindall
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ISSN: 0021-9738 (print), 1558-8238 (online)